Meet Dr. Neetha Joseph- Scientist at NCMR-NCCS Pune

Dr. Neetha Joseph’s research interest is in microbial systematics, ecology and community analysis. She is affiliated with NCMR-NCCS Pune from last 8 years. She is in-charge of FAME analysis service and curator of Firmicutes. It was a great pleasure to interact with Dr. Neetha and to know more about her as a person and her work.

Kranti: Dr. Neetha, you have worked with coastal environment micro-organisms during your PhD. At a personal level, what motivated you to enter into microbiology research?
Dr. Neetha: Kranti, my native place is in Kerala, a beautiful coastal area in India. Kochi is a lovely place with lot of Backwaters and Estuaries. When I finished my post-graduation, I got an opportunity to join at National Institute of Oceanography (NIO) where most of the research work is related to Ocean and Estuaries. Nutrient enrichment due to various anthropogenic activities is the most widespread problem in estuaries around the world. Significant spatial and temporal variability of physico-chemical and geochemical characteristics and productivity patterns are the important characteristics of estuaries. Microbial communities are involved in mineralization of organic matter; therefore, I was interested in understanding the response of these sedimentary microbial communities to these regional and seasonal changes using signature biomolecules (Phospholipid Fatty Acids – PLFA) as a means of identifying the specific group of microorganisms in the natural ecosystems .

Kranti: Everybody has someone in their life who inspires them to achieve something. Who is your inspiration in science?
Dr. Neetha: My PhD guide at NIO, Kochi is my inspiration in Science. She inspired me a lot! She encouraged me in various aspects of science and helped in boosting my confidence.

Kranti: Which methods and tools you use in your research?
Dr. Neetha: Microbial communities are involved in mineralization of organic matter in estuarine sediment. To understand the response of these microbial communities to various physiochemical and geochemical factors using signature biomolecules (Phospholipid Fatty Acids – PLFA) as a means of identifying the specific group of microorganisms in the natural ecosystems. Phospholipids are mainly found in the cell membrane, not in storage lipids and have a rapid turnover in aquatic sediments. So it provides a measure of viable cellular biomass in an ecosystem.  Different physiological and functional groups of microorganisms in sediments were described using PLFA analysis.
The extracted PLFAs were analyzed using gas chromatography (Agilent 7890 Series, USA) with a cross-linked phenyl – methyl siloxane capillary column (25 m, 0.2 mm) and FID. Identification of the FAMEs was carried out by comparison of retention time and equivalent chain length with known standards like Eukary calibration mixture – 1201A (Eukary6 method, Version: 3.7) and MIDI peak identification software (MIDI Inc., Newark, DE).

Kranti: You are contributing to microbiology related services offered at NCMR Pune. What are those services ?
Dr. Neetha: I am in – charge for FAME analysis service and curator of Firmicutes at NCMR. Under FAME analysis, the bacterial (aerobic and anaerobic) or yeast samples are identified based on their cell membrane fatty acids. Also cell membrane fatty acids are analyzed for novel taxa along with their closely related type strains for publication.

Kranti: Are journals necessary in the age of internet? Don’t you think research should be done not just to publish a paper but also to have real life impacts?  
Dr. Neetha: We know that nowadays we can extract all the information we require via internet. But we cannot compare the beauty of reading a book or journal with internet. Yes, I totally agree that we should do research not only to publish a paper but also to have real life impacts.

Kranti: Being a woman in science, what are the challenges that you’ve faced?
Dr. Neetha: Being a woman in science, the major challenge I face is to manage family, children and their education along with my research work. Another challenge is to get time to spend for research along with my routine services and other commitments.

Kranti: How do you maintain the balance of your family and work-life?
Dr. Neetha: For that I should thank my husband and children for their co-operation and moral support throughout my career.

Kranti: What advice would you like to give to young women who want to pursue research?
Dr. Neetha: If you have an actual interest in science along with sincerity, dedication and hardworking nature, you will be able to succeed in your research career. As a woman, you should be able to manage your time and having patience is also equally important to succeed in your life.

Kranti: Would you share with us any memorable incident/moment of your research life?
Dr. Neetha: In the year 2000, I got an opportunity to participate in Cochin – Alleppey – Mangalore Cruise on board CRV Sagar Paschimi, under DOD, COMAPS Programme. It was a rare experience and golden memory in my research life.

Kranti: Most of the scientist’s children opt for career in science. Do you want your child to become a scientist too? 
Dr. Neetha: Yes, if they are showing real interest in science and research, definitely I will encourage him or her to opt for career in Science.

Analysis of SARS-CoV-2 genomes from western India reveals unique linked mutations

Transmission electron micrograph of SARS-CoV-2 (Wikipedia)

COVID-19 is caused by the strain of corona virus named Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2), belonging to the category of betacoronaviruses. The virus mainly causes respiratory illness, varying in severity for different individuals. The COVID-19 pandemic is affecting the whole world. India is one among the worst hit nation by the COVID-19 pandemic. The western part of India is badly affected by the COVID-19 pandemic, the Maharashtra state is a major hotspot for this disease, having around 1/5th of total reported infections in India.

A collaborative research conducted by researchers from NCCS Pune, B. J. Government Medical College, Pune and Armed Forces Medical College Pune present the first comprehensive study on genome and mutation pattern analysis of SARS-CoV-2 from the western part of India. In this study, researchers have investigated the molecular, phylogenomic, and evolutionary dynamics of SARS-CoV-2 in three different regions of Maharashtra, the western state in India. Total 90 genomes were sequenced. The analysis revealed three unique linked mutations which are common in most of the sequences studied. These may act as molecular markers to track the spread of the SARS-CoV-2 virus to different areas.

Nasopharyngeal/throat swabs of suspected COVID-19 patients were collected, samples confirmed with SARS-CoV-2 infection were used for the study. The age of the patients selected in the present study ranged from 2-78, with 80% patients were in the age range of 30-60 years. COVID-19 patient samples with a particular range Ct value for E gene were selected for the genome sequencing. Fast qc tool and BWA (Burrows-Wheeler Aligner) were used for data analysis. Neighbor joining method was used for phylogenomic analysis. Structural and bioinformatics analysis of SARS-CoV-2 variants was performed and comparative study among the Indian samples was also done. The observed mutation pattern was further analyzed to check any relationship with gender, age, and symptoms.

Phylognetic analysis of the genomes revealed that mutations C313T, C5700A, G28881A are unique patterns and observed in 45% of samples, indicating a newly emerging pattern of linked mutations. The Satara district viral strains showed mutations primarily at the 3´ end of the genome, while Nashik district viral strains displayed mutations at the 5´ end of the genome. Characterization of Pune strains showed that a novel variant has overtaken the other strains. Examination of the frequency of three mutations i.e., C313T, C5700A, G28881A in symptomatic versus asymptomatic patients was performed. The analysis showed mutations were prevalent in symptomatic cases, and were more prominent in females. These three mutations were present in more than 30% studied samples of age group 10-25. Interestingly, these mutations were not detected in the higher age group of 61-80.

Study of region-wise mutation pattern among the viral sequences indicated that, a specific pattern of mutation was prevalent in all districts. The relationship of mutation pattern with age, gender and symptoms was studied. A distinct pattern was observed in age-wise distribution, some of the mutations were prevalent in the age group of 10-25. The proportion of three mutations C313T, C5700A, G28881A were found relatively higher (~80%) in symptomatic patient samples as compared to asymptomatic (40-50%). Also, the mutation C241T was found in 90% of all the sequences and is located in the 5′ UTR region and found predominantly in severely affected patients. However, the role of this mutation has not been studied yet.

The comparative study indicated that, distinct sub-clones of virus were prevalent in different parts of India at the same time period. The type 19A clade virus was predominant in Delhi (Northern part) whereas in Maharashtra (western part) 20A, 20B clade virus was dominant in April-May 2020. While in Telangana (southern part), 19A clade was dominant in April, and it shifted completely to 20A and 20B in May 2020. Because of lockdown, factors contributing to transmission of SARS-CoV-2 virus was restricted. The researchers are assertive about prevalence of a specific viral variant in a region could be attributed to human host susceptibility for specific viral variants. This susceptibility seems to be based on mutations prevalent in the viral variants in that region.


Pea plant shapes its rhizosphere microbiome for nutrient uptake and combating stress

-By Kranti Karande

Legume crops like Pea are used as rotation crops along with rice cultivation in long term conservation agriculture experiments in the acidic soils of the North East region of India. Rhizosphere microbiomes present in the soil have significant influence on plant growth and productivity. The study aims at understanding the bacterial composition of microbiomes present in bulk soil as compared to the rhizosphere. It also aims to understand how the pea plant influences the bacterial communities present in soil and the rhizosphere microbiome in order to improve nutrient uptake and stress improvement. Pea cultivation is a practice used in conservation agriculture which strives to preserve and enrich the environmental resources to sustain and improve crop productivity. The study conducted will help devise future strategies to expand pea cultivation and improve soil health in the region. 

Crop rotation is an important and effective strategy as part of conservation agriculture practices. The North East region of India is a fragile, marginal, inaccessible and diverse ecosystem. Generally a mono-cropping system of rice is followed in this region.  Zero tillage (without disturbing the soil) cultivation of pea (Pisum sativum L.) has been considered beneficial to enhance the cropping intensity in the region. The majority of soils in North-East India are acidic in nature. The pH of soil among many other environmental factors has a significant influence on the type of nutrients and microorganisms present in the soil which in turn have an influence on the productivity of crops. Similarly, nutrient and residue management practices like the application of chemical fertilizers often influence the endogenous microbial communities. 

Sample collection for the study was done from experimental fields of the ICAR Research Complex for NEH Region, Umiam, Meghalaya, located in Eastern Himalayan region. Different tillage and residue management treatments were maintained in these fields for the last eight years by alternatively cultivating rice followed by pea cultivation. For microbial community analysis, bulk soil and pea rhizosphere samples were collected from each treatment plot. All the samples were processed for community DNA extraction. Analysis of the chemical properties of the soil samples was done using available methods. Rhizosphere soils were harvested from roots of pea plants. 

Soil pH (1:2.5) was found to be influenced by tillage and nutrient management practices at depth  0-15 cm. The combined effect of tillage and nutrient management practices on available N, P and K content and SOC,TOC of soil were significant. The rhizosphere showed higher diversity indices in comparison to the bulk soil samples. A total of 71 bacterial phyla were detected in the bulk soil and rhizosphere samples. A higher abundance of Firmicutes was recorded in bulk soil (~41.7%) in comparison to the rhizosphere (~17.8%). On the contrary, Proteobacteria were highly abundant in the rhizosphere (~43.9%) in comparison to bulk soil (~18.6%) samples. Significantly higher abundance of Proteobacteria and Bacteroidetes was observed in pea rhizosphere samples in comparison to bulk soil. 

Impact of residue management practices on abundance of specific microbial communities was observed across both rhizosphere and bulk soil samples. The impact of tillage history was also observed on the enrichment of specific OTUs in the bulk soil and rhizospheric soil. Differences in the abundance of 11 genera were recorded in the rhizosphere sample across the history of different tillage treatment. All these genera showed higher abundance in the conventional tillage fields. The correlation between soil properties and microbial community structure was also studied as part of the study. Significant correlations were observed between relative abundance of few bacterial phyla & genera and soil properties in both bulk soil and rhizospheric soil samples. However, the number of significant correlations was low in rhizosphere samples, in comparison to bulk soil samples. 

The study was designed to investigate the effect of long-term exposure to various tillage and residue management practices on the bacterial community structures of the bulk soils and how pea plant (a rotation crop) shapes the rhizosphere communities. A higher species diversity and evenness was observed in rhizospheric samples. There was no significant difference in bacterial richness and evenness among different tillage and residue management treatments in both rhizospheric and bulk soil samples. This is an indication that the plant rhizosphere effect (a plant’s ability to alter microbial communities in rhizospheric soil) is the key driver of alpha diversity. Plants can alter the microbial communities by secreting a variety of nutrients and bioactive molecules into the rhizosphere. Enrichment of specific OYUs in the Pea rhizosphere were also confirmed which can be attributed to the selection pressure of the Pea root. The results of the Pea rhizosphere and bulk soils were consistent with the fact that the majority of members of microbial communities in the host plant are horizontally acquired from the surrounding environment, and the soil is the main reservoir of a plant rhizosphere microbiome. The genus Nitrobacter was at higher abundance in pea rhizosphere samples than bulk soils, suggesting its enrichment by the host plant as Nitrobacter converts nitrite to nitrate making nitrogen more readily available to the host plant. Higher abundance of genes related to nitrogen fixation, phytohormone and siderophore production, phosphate solubilization in the rhizosphere soil substantiate the conclusion that the selection of bacterial communities is always based on plant growth promoting potential in the rhizosphere. 

The study concluded that pea plant is the most dominating selection factor shaping the microbial communities under diverse residue management and tillage treatments. The rhizospheric soil was found to be enriched with bacterial taxa known for plant growth promotion which indicates that the plant plays a role in selecting the rhizospheric communities to meet its requirement of nutrient uptake and combating stress.


Identification of pathogenic yeasts from different clinical samples

-By Kranti Karande

Yeasts have always been a part of human microbiota. Some of the species belonging to the yeast family are opportunistic pathogens leading to infections of cutaneous, mucosal, bloodstream or deep-seated organs known as Candidiasis. These infections have become a major threat to humans. Different species of the Candida genus lead to different medical conditions and demonstrate varied sensitivity towards anti fungal agents used in practice. Failure to commence accurate anti-Candida therapy at an appropriate time has led to an increase in fatal cases of Candidiasis. Hence, it is important to correctly identify the Candida species to start appropriate and timely treatment. There are very few studies of yeast infections in Indian context. Thus this study is important as it is a study of a large number of Indian clinical samples for yeast infection reporting opportunistic and emerging pathogens. The study conducted on 176 clinical samples collected from Bharati Hospitals, Pune, Maharashtra was aimed at identifying the pathogenic species of yeast, understanding their anti fungal susceptibility and cell invasion capabilities.

Existing techniques for species identification of Candida species may be time consuming and even resulting in non-authentic identification if carried out by classical usage of blood culture technique. Recently developed molecular tests provide real time PCR assays and rapid results. Although these methods are specific, they do not improve the diagnostic sensitivity of Candidaemia and Candidiasis. Nonetheless DNA sequencing of amplified PCR products continues to be the most reliable method for authentic fungal species identification. A comparison of three different techniques for yeasts species identification was carried out along with characterization of these species for antifungal susceptibility and cellular invasion capabilities.

The clinical samples obtained were chemically treated as per standard protocol before being microscopically examined to find out the presence of unicellular yeast cells and other yeast organs. Samples with probable yeast were further treated to obtain isolated colonies and consequently obtain pure cultures which were deposited at NCMR- NCCS Pune, India. The colonies were further characterized biochemically using different identification tests. 

PCR amplification, DNA sequencing and phylogenetic analysis:
Genomic DNA from each isolate was extracted using lithium acetate. The precipitated DNA was used for PCR amplification (increasing the copies of DNA) using suitable primers. After confirmation of amplification, the PCR product was sequenced to identify different species present in the samples. A phylogenetic tree was constructed individually for each strain to confirm its species identification using known sequences of different Candida species available in GenBank at NCBI. 

MALDI-TOF/MS biotyper and chromogenic media method:
Actively growing pure cultures were used for protein extraction for MALDI-TOF MS analysis. The extracted protein samples were analyzed using a 60 Hz Nitrogen Laser and Flex Control software. Chromogenic media screening of yeasts was also performed and identification of colonies was done using colony color and appearance. Chromogenic media is used commonly in clinical laboratories for rapid identification of Candida strains. It is a fast method but not entirely reliable. Test results showed that chromogenic media successfully identified only 64 out of the 75 strains identified as C. albicans by sequencing. Seven strains were misidentified and four remained unidentified. Hence, it is safe to say that chromogenic agar technique is not a reliable method for yeast species identification. 

A total of 176 isolates were analyzed using MALDI-TOF/MS and the results were compared with results of DNA sequencing. MALDI-TOF MS could identify 157 of yeast isolates correctly. Overall, the correct identification rates of the 176 yeast isolates to species levels by the Bruker MALDI Biotyper systems was 89.2. This study confirms that MALDI-TOF MS presents an effective alternative to the sequencing for the correct identification of the emerging yeast pathogens however, there is a need to improve the database by regularly adding newer yeasts species from clinical and environmental scenarios.

Invasive fungal infections are becoming common and its rapidity of invasiveness by pathogens demands early arrest of infection by antifungal agents. There are immense changes in the host factors, infecting fungi, and antifungal agents and hence there is a need to accomplish antifungal susceptibility tests. The study conducted involved characterization of yeast species in terms of their antifungal susceptibility. A total of 157 isolates were tested for their susceptibility to antifungal drugs. Clinical yeasts showed increased resistance to fluconazole (55%) which is the most common antifungal used for treatment of candidiasis. Some emerging pathogens were found to be sensitive to most of the tested antifungal agents. It has been observed that strains of C. glabrata have highest resistance among various species of Candida. Many researchers have also isolated multi-drug resistant strains of C. glabrata

In vitro cell invasion assay involved investigation of the ability of various yeasts isolates to invade epithelial cells (HeLa Cells were used for this study). Since the invasion of host cells by pathogenic yeast cells is one of the important virulence factors in invasive candidiasis, this study is significant for the purpose of understanding potential threats from each Candida species. A total of 88 yeast isolates tested were isolated from invasive Candidiasis patients and only few could show invasion in vitro. Although the results of invasion capabilities study for different species like C. albicans, C. glabrata, C. parapsilosis showed varied results, it was observed that yeast cells invade host cells with the help of pseudo-hyphae or hyphal structures. Some studies have also shown that the transformation of C. albicans into hyphal form increases its interaction with the tissue cells, thus increasing its ability to adhere to human cells and potent invasion.

MALDI-TOF MS is an important tool in clinical set up for microbial identification as it is fast, low-cost, simple to use and wide spectrum applications in the identification of bacteria, archaea, and fungi. However, there is an urgent need to identify the species directly from the sample since it will greatly save on time of treatment which is crucial in serious cases like invasive Candidiasis. At present, both the methods which are considered reliable require pure culture, i.e. DNA sequencing and MALDI-TOF MS. The antifungal susceptibility tests showed that a large number of isolates showed resistance to all antifungals tested. A molecular analysis of all these strains is essential to substantiate the cause and mechanism of resistance. In the present study, nystatin was found to be the most effective antifungal agent. Many new types of yeast were reported in the study as probable human pathogens which should be included in diagnostic protocols.


Description of a novel species isolated from the surface of tomato

-By Kranti Karande

A new bacterial strain designated as TOUT106T was isolated from the tomato surface. Though there were many bacterial species isolated from various fruits and vegetables like sweet potato, banana, tomato, lettuce and cucumber, TOUT106T was found to represent a novel species. This study was aimed at providing a detailed taxonomic description of this novel strain TOUT106T isolated from tomato surface. A novel strain is a micro organism which hasn’t been identified earlier.

The strain was isolated by washing the outer surface of a tomato collected from a local vegetable market in Pune, India. Colonies grown on a trypticase soy agar turned out to be 1-3 mm in diameter, circular, raised with an entire margin, and translucent opacity. The identification was done using MALDI-TOF MS technique. After extracting high quality genomic DNA from the strain, the 16s rRNA sequence was amplified. The similarity search for the 16S rRNA gene sequence of strain TOUT106T was performed against the type strains of prokaryotic species in the EzBioCloud’s database. 

Genome sequencing was performed and from the concatenated sequences of 92 core genes extracted, a possible phylogenetic tree was inferred. The Average Nucleotide Identity (ANI) was determined between strain TOUT106T and closely related strains of the Enterobacteriaceae family. Analysis of chemotaxonomic features (based on fatty acids and cell proteins)was done after harvesting cell biomass from culture grown on TSA at 28°C.  Antibiotic susceptibility was determined using the disc diffusion method. The susceptibility to antibiotics was interpreted based on the Clinical and Laboratory Standards Institute (CLSI) guidelines determined for members of the family Enterobacteriaceae

A search of the 16S rRNA gene sequence of strain TOUT106T showed the highest similarity to Salmonella enterica subsp. arizonae strain NCTC 8297T (98.4 %) . The phylogenetic tree constructed based on the 16S rRNA gene sequence placed the strain TOUT106T within the Salmonella clade. The genomic DNA content of strain TOUT106T was well within the specifications of genus Klebsiella. Comparative analysis of ANI value and dDDH relatedness of Enterobacteriaceae strains suggested that the strain TOUT106T is a novel species.  A comparison of MALDI-TOF MS spectra based dendrogram showed that the strain TOUT106T separated from the type strains of Salmonella and was placed along with Klebsiella variicola DSM 15968T and Raoultella terrigena DSM 2687T, corroborating well with the results of genome-based analysis. Colony morphology, as examined on blood agar medium, were mucoid and translucent.

Based on different methods of identification, scientists reported that the strain TOUT106T is a member of genus Klebsiella. However, it differs from closely related species of the genus Klebsiella in several aspects, such as biochemical features, physiological features, protein profile, and overall genome relatedness indices. Thus, it represents a novel species in the genus Klebsiella, for which the name Klebsiella indica sp. nov. is proposed.

Klebsiella indica (in’ L. fem. adj. indica, of or belonging to India, where the type strain was isolated from the outer wash of a tomato collected from the vegetable market in India). Cells are Gram-negative, straight rods with round ends (0.7-0.9×2-3 μm), and non-motile. Colonies grown on trypticase soy agar are 1-3 mm in diameter, circular, and with translucent opacity. The optimal temperature for growth is 28 °C, and the optimal pH is 7.0. Growth occurs in the absence of NaCl with up to 2% tolerance in trypticase soy broth. It is weakly positive for catalase and negative for oxidase activity. It is susceptible to the majority of antibiotics. 

Reference :

Microbiome of indian patrilineal families reveal association with age

-By Kranti Karande

The human microbiome plays an important role in maintaining stable health conditions. It is influenced by age, geography, diet and other factors. This study was aimed at understanding the association of composition of the human microbiome with age in Indian joint families formed through paternal descendants. Oral, skin and stool microbiome of a total of 54 healthy individuals from 6 joint families with three generations were studied and characterized using 16S rRNA gene based methodology. The study population had matching dietary, social habits, hygiene and sanitation habits, economic status and geographic position.  This study highlights that precise and perceptible association of age with microbiome can be drawn when other causal factors are kept constant. 

Human microbiome has evolved with the host and its ancestors for millions of years and it plays an important role in maintaining a good health by performing various functions such as digestion, protection against pathogen colonization to host immunity and regulation of central nervous system. The human microbiome is affected by various factors such as ethnicity, age, diet etc. Hence, it is important to study the same population for the exploration of a precise association of age and microbiome. This study of genetically linked individuals of different generations having similar diet, ethnicity and location will help to understand the ability of microbiome to persevere with increasing age and how they progress with the age. 

Approximately 99% of the gut microbiome was constituted of 5 bacterial phyla. Total of 174 bacterial genera were noted to be present out of which 5 contributed to 77% of the gut microbiome. The oral microbiome showed comparatively higher abundance of some specific bacterial phyla.  Bacterial genera prevalent in 95% of the study population with more than 0.1% abundance were considered as a part of the core microbiome. Gut, Oral and skin microbiome had 3, 13 and 2 core microbiome genera present in the samples respectively. 

Microbiome community structure of gut, oral and skin samples was investigated across three generations (age groups). Gut microbiome of each generation had a unique set of bacterial genera present in abundance out of the prevalent genera for the specific age group. High abundance of few bacterial taxa was recorded in particular age groups in the skin microbiome samples also. Comparative microbiome analysis in three age groups did not show significant difference in abundance of bacterial genera in the gut and skin microbiome. However, the oral microbiome showed significant variations in the abundance of genera Dialister, Fusobacterium, Streptococcus, Selenomonas, Filifactor and Treponema. 

Age-associated changes in the microbiome were further analyzed based on differentially abundant OTUs (a methodology). After performing a correlation analysis it was revealed that phylum Proteobacteria in gut microbiome and phylum Fusobacteria in oral microbiome showed higher abundance with increasing age. However, in the skin microbiome, no such statistically significant correlations were noted. Amongst the total 171 bacterial genera in the gut microbiome, only genus Bacteroides showed age-associated changes. Decreased abundance of Bacteroides was noted with increasing age.

Dietary information of the study population was collected using the food frequency questionnaire (FFQ) and this information is subsequently translated into the daily intake of carbohydrates, proteins, fats, lipids, fibers and calories with the help of a nutritionist. Detailed analysis showed that carbohydrates provide about 74%, 81% and 80% calories in the first, second and third generation members, respectively. Further analysis showed no significant correlation across generations suggesting similar microbiome structure and dietary pattern. This emphasizes the fact that overall homogeneity in the diet helps in maintaining the microbial state.

Bacteria with high fiber degrading potential were found highly abundant in first generation members while the second generation members showed an abundance of metabolism boosting gut microbiome. Early gut colonizers and Bacteroides were higher in the third generation members. The skin microbiome also showed age related changes in abundance of bacterial taxa present. With the increasing age, physiological changes occur in the skin structure which explains the association of key bacterial taxa in the members of the respective age groups. Similarly, in the oral microbiome, Fusobacteria was found to increase with increasing age. It was observed that a negative correlation in the abundance of Bacteroides with age; this is in contrast to previous studies demonstrating the higher abundance of genus Bacteroides with increasing age. Age related changes in oral microbiome could be associated with physiological changes occurring with increasing age in the oral cavity. 

In conclusion, this study particularly highlights the precise and perceptible association of age with the microbiome. The findings suggest that core taxa constitute more than 75% of the gut and oral microbiome, while only 67% of the skin microbiome, indicating a larger variability of the microbiome present on the skin. The baseline data presented from a healthy Indian sub-population can be used as reference for further studies including diabetes, obesity and inflammatory diseases. 


Environmental and public health perspective of WasteWater Treatment Plants (WWTP)- safe or unsafe?

-By Kranti Karande

The population of Pune city located in the state of Maharashtra, India is around 31 lakhs. Imagine how much waste water such a huge number of people produces!  The amount of wastewater produced at Pune City is approximately 744 MLD (millions litres per day). Out of this, the WasteWater Treatment Plants (WWTP) treats about 527 MLD of water. The waste water treatment plants aim to minimize the adverse effect of untreated wastewater on ecology, the environment and human health through its adequate treatment before release into the ecosystem. But the question is, is this treated water indeed safe for environmental and public health? Whether or not the microorganisms present in the untreated water are eradicated following treatment? How WWTP contributing in spread of antimicrobial resistance (AMR) in nature?

Dr. Om Prakash Sharma’s group from NCMR, NCCS Pune started with an interesting question as to whether there is a difference between the number of bacteria in the sample of untreated and treated waste water. Different types of antibiotics are used in human therapy, veterinary and animal farming, and huge number of antibiotics are released into municipal wastewater which ultimately finds its way into the environment. Dr. Sharma’s group also tried to answer the question of whether or not the wastewater-isolated bacteria develop antimicrobial resistance.

They collected samples from untreated and treated waste water and stored it at appropriate storage conditions for further processing. The bacterial number was estimated by total viable count and most probable number methods and efficacy of current wastewater treatment plant in reduction of bacterial load and spread of antibiotic resistant bacteria in the environment was also studied.

A total of 30 bacterial species belonging to 18 different genera were isolated from the untreated water sample. Whereas in the treated wastewater only 9 species from 6 different genera were present, all 9 species of bacteria are non-pathogenic. They also performed antimicrobial susceptibility  testing using  these bacteria to know the role of WWTP in spread of AMR. Twenty one antibiotics have been used for testing. All of these antibiotics are common in testing antimicrobial resistance in bacteria. The bacteria isolated from untreated water samples indicated high levels of antibiotic resistance.

The research group concluded that, untreated wastewater sample contained wide range of organisms with high levels of antibiotic resistance while bacterial load reduced drastically and pathogens were absent in the treated wastewater. Results indicated that the wastewater treatment plant was working effectively and efficiently by reducing the bacterial load in treated wastewater. Several organisms of clinical significance like Acinetobacter septicus, Citrobacter farmeri, Klebsiella oxytoca, Raoultellao rnithinolytica etc. were also reported in untreated wastewater sample.

This study demonstrated the comparison between culturable bacterial population present in influent and effluent of municipal waste water treatment plant of Pune. The researchers conclude that waste water treatment not only reduces BOD (Biochemical Oxygen Demand), COD (Chemical Oxygen Demand) and TSS (Total Suspended solids) but also bacterial load of the waste water.


Meet Dr. Rohit Sharma: Mycologist at NCMR, NCCS Pune

Dr. Rohit Sharma is a fungal taxonomist working at NCMR, NCCS Pune. He has been working in this field from last 15 years. He has identified 2 novel genera and 11 novel species of fungi. It was a great pleasure to interact with Dr. Sharma and find out more about him and his work.

Kranti: What motivated you to enter into the research field of fungal taxonomy?
Dr. Sharma:
I think it all started quite early when I used to go for sampling with my father, a Plant Pathologist at J.N. Agriculture University, Jabalpur. I used to go on collection trips with him during my school and college days to collect fungi infecting crops and wild plants and initial interest developed from there. During this period, I also got an opportunity to meet Dr. Kalman Vanky, expert in smut fungi and got opportunity to see his dedication towards the field of smut taxonomy. However, the actual involvement started when I began my doctoral work under Dr. Akhilesh K . Pandey and Dr. Ram C. Rajak whose lab was known to work on diversity and taxonomy of fungi. Here, I could get experience on entomopathogenic fungi, pathogenic and saprophytic micro fungi, mushrooms and yeasts. I developed a lot of insight in the lab under their guidance. Regular interactions helped to understand basics and the lab was rich in literature (monographs, journals and manuals) which usually is a bottle neck in the morpho-taxonomy of fungi. In the present organization as a curator of fungi, I got an opportunity to handle many fungi (yeasts, mycelial fungi and mushrooms) that helped in the development of the expertise in fungal taxonomy and systematics including polyphasic taxonomy.

Kranti : Why it is important to study fungal taxonomy?
Dr. Sharma: Fungi are hyper-diverse group of organisms. So far, many have been discovered but many are still to be discovered. Till now, about 1,20,000 fungi are known and researchers are discovering many more with an annual rate of approx. 1000 fungi. Based on the environmental sequence/ metagnomic data, it is now estimated that 22-38 lakhs fungi are yet to be discovered which makes it important to study the fungal diversity and taxonomy. Moreover, since they play an important role in environment and industrial biotechnology, it is important to explore, identify and subsequently study for their bio prospecting. Moreover, human and plant pathogens are increasing day by day and many environmental opportunistic pathogens are causing infections. Hence, it becomes important to have their authentic identification and proper classification for better management. The fungal taxonomy is complex and includes species specific morphological and physiological characters and intraspecific variation. The DNA-based taxonomic studies have helped to resolve many taxonomic problems and describe several cryptic species and are considered more stable than morphological characters.

Kranti: How many fungi you have collected and identified till now?
Dr. Sharma: So far we have collected and identified more than one thousand fungi from Lonar lake-Maharashtra, Famlong Lho-Sikkim, Achanakmar Sanctuary- Chhattisgarh, and other sites of India. They have been isolated from soil, litter, insect gut, sediments, water and as plant endophytes. We at NCMR-NCCS have described 2 novel genera (Matsushimamyces and Alanomyces) and 12 novel species, viz., Naganishia indica, Coniochaeta dendrobiicola, Leucosporidium himalayensis, Aureobasidium tremulum, Matsushimamyces bohaniensis, Alanomyces indica, Nothophoma raii, Curvularia lonarensis, Arthrinium gutiae, Pyrenochaeta telephoniae, Chaetomium jatrophae and Arthrinium jatrophae.

Kranti: What is your contributing role in services at NCMR?
Dr. Sharma: At present, I have been working as Curator-Fungi and looking after more than 15,000 fungal cultures preserved at our collection. My earlier work mostly was related to development of fungal culture collection at NCCS-NCMR, development of protocols, training human resource and undertaking basic research. So far, we have processed more than 2500 fungal cultures deposited by researchers from academia and from industry and accessioned approximately one thousand of them. We also provide identification services by conventional (morphology and API kit) as well molecular method, customized services and contractual research to industries. I along with my technician and project staff have been able to deposit more than 500 cultures to the NCMR-NCCS culture collection and screened them for various potential industrial applications viz., enzymes, bio active metabolites, waste degradation, etc. During the period, I have handled 4 research projects focusing on fungal diversity and their bio prospecting which in turn contributed to enrich the culture collection and preserve fungal wealth of our country.

Kranti: How has your journey been from being a PhD student to a Scientist at prestigious national institute?
Dr. Sharma: It has been a nice learning process, from working on ectomycorrhizal mushrooms during doctoral thesis to working with micro-fungi and yeasts at NCMR-NCCS. It has state of the art facilities and I can now use advanced techniques for my work. Work culture is very nice in our institute and  freedom to work and pursue my research interest in the area of diversity and taxonomy.

Kranti: What are the challenges you face during a field trip?
Dr. Sharma: During my doctoral thesis, I have went for sampling in the forests of Madhya Pradesh and Chhattisgarh for collection of ectomycorrhizal mushrooms. There are several challenges faced due to lack of communication, remoteness of the forests, etc. Moreover, we used to collect mushrooms from 8 am in the morning to 5 pm in the evening with a small packed lunch. In the evening after arriving at the forest guest house, we used to complete the mushroom data sheets and then around 7-pm we used to start culturing the mushrooms before putting them for drying. Sometimes it used to get 2-3 am in the morning because the mushrooms might decompose as the time passes after their harvest from the field.

Kranti: Which is your favorite fungus?
Dr. Sharma: The whole group of fungi are fascinating and one cannot pinpoint a single fungus. These are morphologically diverse and each one is unique in their features viz., colony color, shape, conidial shape, size, etc. The sexual and asexual stage found in these groups of organism makes them more interesting. From microscopic mycelial fungi to multi-cellular mushrooms to single celled yeasts, all have uniqueness of their own. From exogenous spores to endogenous spores, from pycnidia to cleistothecia, all have different morphological structures.

Kranti: What are the storage methods used at NCMR?
Dr. Sharma: We at NCMR-NCCS preserve the culture by five methods viz., cryopreservation at -80oC and at -196oC (liquid nitrogen) and by freeze drying. Apart from this, we also preserve the fungal cultures in distilled water and mineral oil (at 4oC). 

Kranti: What is your support system?
Dr. Sharma: My family and friends are my support system at individual level and colleagues and my team working with me at the professional level.

Kranti: What would you have liked to become if not a scientist?
Dr. Sharma: I would have been involved in doing agriculture, preferably doing scientific and organic agriculture with little bit of teaching at some academic organization so that I can interact and dissipate knowledge to students.

Kranti: What are the current and future projects in your group?
Dr. Sharma: Currently, we are working on collecting fungi from various sites and bio prospecting them for various applications like antimicrobial activity and treatment of industrial effluent. In future, we would like to focus more on the industrial applications of the fungal resource preserved in our collection and develop biotechnological usage of them.

Kranti: What are you hobbies?
Dr. Sharma: I like to read general books. Gardening is one my favorite hobbies and like to grow flowering as well as vegetable plants.

Kranti: Does research sometimes becomes stressful? What do you do for relaxation?
Dr. Sharma: Yes, research sometimes is stressful as sometimes multiple things gets lined up at the same time. To relax, I listen to classical music or go for trekking at nearby Pashan Tekri.

Kranti: What are your thoughts on Science communication in India? How can scientist contribute for better Science communication?
Dr. Sharma: I remember during my childhood I used to read a magazine ‘Science Reporter’, it used to explain scientific discoveries in simple language. Science communication in India is developing over the years by means of audio-visual and writing medium. Scientists can contribute for better science communication by conducting science talks for undergraduates explaining their research in a simplified manner. It is better now than it used to be. There are more communication channels available now with online platforms like blogs and online articles as compared to earlier days. 

The Importance of Studying Soil Microbes and their Interactions

-By Kranti Karande

A large number of micro-organisms are present in the soil ecosystem. There is a popular phrase that, microbes in a handful of soil are more in number compared to the total number of human beings that have ever existed on this planet. Soil life consists of microbes, nematodes, earthworms, ants, other insects, etc. With an estimated 100 billion bacteria that can be found in a handful of soil, it is the largest group of organism in this life-sustaining ecosystem. It is an astonishing reality that this handful of soil contains about 500 individual species of fungi and its mycelium can run up to 50 km in length. Staggering, isn’t it!

Among microbes, bacteria are present in large number in soil. Soil bacteria are mainly responsible for nitrogen fixation. Actinomyecetes, a group of bacteria break large lignin molecules into small molecules. Although not as commonly abundant as bacteria, fungi also significantly contribute in soil health by decomposing organic matter and nutrient recycling. Microbes contribute to soil in various ways by increasing its fertility, aggregation ability and by fixing nitrogen.

Soil ecosystem plays an important role in cultivating high yielding crops. Microbes being the major community of the soil, it is important to study them in order to conserve and nurture soil ecosystem. Studying soil microbes will help in increasing soil fertility and indirectly will contribute for betterment of farmers’ lives and for betterment of society.

Isn’t it interesting to study how these tiny microbes contribute in overall plant health? The very first interaction between plants and microorganisms occurs in soil. Every plant is associated with a unique rhizosphere (root microbial community). The rhizosphere microbial community is selected from large number of microorganisms present in the soil. The symbiotic association (beneficial for both) between rhizosphere and plant leads to complex interactions contributing to plant growth.

The interactions between these three components: plants, microbes and soil system play a critical role in maintaining health of the plant. However, the complexity of these interactions is not yet clear. Research group led by Dr. Kamlesh Jangid at NCMR, NCCS Pune is trying to understand this complex interaction.

Animals call for help when in need, but can you imagine how bacteria might be communicating with each other, especially in the complex soil ecosystem? You may wonder whether they use mobile phones. Not really, but they do have a very advanced communication system. Bacteria produce and release signalling molecules called as Auto inducers (AIs), which are then sensed by neighbouring organisms enabling them to differentiate between self and non-self. Isn’t it fascinating? This mechanism of cell-to-cell communication is known as quorum sensing.

While the mechanism was first discovered in 1970s by the team of Kenneth Nealson, Terry Platt and J. Woodland Hastings, it was not until 1994 that the term “quorum” was associated with this density-dependant mechanism by Fuqua , Winans and Greenberg. Quorum is a mechanism by which bacteria plan and fine-tune their actions as a group rather than as individual cells, thus co-ordinating gene expression and overall microbial population behaviour. Dr. Jangid’s group is discovering the presence of this mechanism across the bacterial community in soils. Studying this mechanism will help the scientific community to better understand the gene regulation in the soil microbes and will answer the questions related to their functional roles.

While studying the most abundant bacteria in soil, affiliated with the phylum Actinobacteria, Dr. Jangid’s group discovered that quorum sensing is extremely under explored partly due to the lack of sensor systems that can detect the huge diversity of AI molecules secreted by this group of bacteria. Specifically, only nine out of the 342 genera in the phylum Actinobacteria are experimentally proven to have this communication mechanism. Dr. Jangid’s group is now developing sensor systems for detecting signalling molecules produced by these bacteria to better understand quorum sensing in this phylum. This research can likely contribute to agricultural, biotechnological, medical and ecological fields.

If we understand the distribution of quorum sensing in soil bacteria, we will be able to modulate soil communities to enhance soil health and increase overall crop productivity. In addition, this will facilitate our understanding of the communication between microbes present in the rhizosphere and why plants are associated with a unique rhizosphere (root microbial community).

It was a great pleasure to interact with Dr. Jangid. In the discussion, Dr. Jangid commented that, “Soil microbiology and bacterial quorum sensing are two separately followed niche fields and their interjection enables us to explore new paths for improving soil health and creating more sustainable agricultural practices. Quorum sensing is also being researched extensively for developing new synthetic analogues that block (or quench) this communication mechanism among pathogenic microbes rather than the conventional anti-microbial drugs in use. The advantage of quorum quenching is that unlike antimicrobial targets, we expect very little to none resurgence towards this approach”.

Link to Dr. Jangid’s lab webpage:

Dr. Kamlesh Jangid with his research team

First Phytoplasma whole genome sequence from India

-By Kranti Karande

Phytoplasma is a group of extremely small bacteria. They don’t have a cell wall and any particular shape. Phytoplasma was first identified by a Japanese scientist Yoji Doi as ‘mycoplasma-like-organisms’ in 1967. They are bacterial parasites of plants and insects. 

Sugarcane Grassy Shoot (SCGS) is a disease caused by phytoplasma and the disease leads to 5% to 20% crop loss. SCGS phytoplasma causes severe proliferation of tillers leading to the typical grassy appearance of sugarcane, hence the name grassy shoot is given. Another disease called Bermuda Grass White leaf (BGWL) disease is a destructive phytoplasma disease of Bermuda grass.

Research group of Dr. Amit Yadav at NCMR, NCCS Pune performed whole genome sequencing of two phytoplasma associated with these two diseases. This is the first whole genome sequence of phytoplasma published from India. This sequence data might help in taxonomical characterization of other phytoplasma belonging to the same group.

A sugarcane plant sample (strain SCGS) exhibiting grassy shoot symptoms and a Bermuda grass sample (strain LW01) showing white leaf symptoms were collected from Pune, Maharashtra, India. The presence of phytoplasma was confirmed by 16S rRNA gene sequencing. Researcher confirmed that these phytoplasma strains belonged to the 16SrXI and 16SrXIV phytoplasma groups, respectively. Both genomes were annotated using the NCBI Prokaryotic Genome Annotation Pipeline

The final SCGS assembly contained 29 scaffolds corresponding to 505,173 base pairs of DNA. The genome was found to be 95.43% complete and the GC content was 19.86%.The SCGS genome was predicted to have 404 protein-coding genes, 12 tRNA and two rRNA genes.

Similarly, the LW01 assembly contained 21 scaffolds corresponding to 483,935 base pairs of DNA. This genome was found to be 91.32 % complete and the GC content was 20.46 %. The LW01 genome was predicted to have 425 protein-coding genes, 13 tRNA and three rRNA genes.